MHC molecules exist in two forms, class I and class II, both encoded within a single gene complex. MHC genes are highly polymorphic: most loci have up to about 100 alleles in the human population (Hansen, T. H., et al. 1993 In “Fundamental Immunology” Ed. Paul, W. E., RavenPress, New York, N.Y., p. 577).
Class I MHC molecules are 45 kD transmembrane glycoproteins, noncovalently associated with another glycoprotein, the 12 kD beta-2 microglobulin. The latter is not inserted into the cell membrane, and is encoded outside the MHC. Human class I molecules are of three different isotypes, termed HLA-A, -B, and -C, encoded in separate loci. The tissue expression of class I molecules is ubiquitous and codominant. The three-dimensional structure of several human and murine class I molecules have been resolved (Bjorkman, P. J., et al. (1987) Nature, 329, 506; Garrett, T. P. J., et al. (1989) Nature, 342, 692; Madden, D. R., et al. (1991) Nature, 353, 321; Fremont, D. H., et al. (1992) Science, 257, 919). Their first and second extracellular domains fold into a binding site consisting of a β-pleated sheet floor flanked by two parallel α-helical portions. The binding site presents 7-9 amino acid long antigenic peptides to cytotoxic effector T lymphocytes (Tc) (Madden et al. and Fremont et al., above). Most of these peptides arise from proteins synthesized inside the antigen presenting cell (APC), e.g., from proteins of viruses or other intracellular parasites, or from misfolded self proteins. The three class I isotypes, as well as their allelic forms, have different peptide binding specificities, depending on polymorphic residues within the binding site (Falk, K, et al. (1991) Nature, 351, 290; Falk, K, et al. (1992) Eur. J. Immunol., 22,277). There is an additional binding site on the third class I domain that interacts with CD8 molecules expressed selectively on Tc cells. The initial step in Tc cell activation is the simultaneous interaction of their antigen receptor (TCR) with the presented peptide and CD8 with its acceptor site on the same class I molecule.
Class II MHC molecules are noncovalently associated heterodimers of two transmembrane glycoproteins, the 35 kD α chain and the 28 kD β chain. In humans, class II molecules occur as three different isotypes, termed HLA-DP, -DQ, and -DR. Polymorphism in DR is restricted to the β chain, whereas both chains are polymorphic in the DP and DQ isotypes. Class II molecules are expressed codominantly, but in contrast to class I, exhibit a restricted tissue distribution: they are present only on the surface of cells of the immune system (constitutive expression on B lymphocytes and dendritic cells, and inducible expression on T cells and monocytes). The three-dimensional structure of three different DR molecules has been determined (Brown, J. H., et al. (1993), Nature, 364, 33; Stern, L. J., et al. (1994) Nature, 388, 215; Ghosh, P., et al. (1995) Nature, 378, 457; Dessen, A., et al. (1997) Immunity, 7, 473). Overall, their structure is very similar to that of class I molecules. The peptide binding site is composed of the first domains of α nd β chain, which, in contrast to class I, is open on both sides, allowing the binding of longer (12-24 residues long) peptides (Chicz, R. M., et al. (1992) Nature, 358, 764). An additional binding site on the second domain of β chains interacts with the CD4 molecule, expressed selectively on helper T (Th) cells. This molecule has a co-receptor function for Th cells, analogous to that of CD8 for Tc cells. During their biosynthesis and intracellular transport, class II heterodimers are chaperoned by a third, nonpolymorphic non-MHC-encoded 31 kD protein, termed invariant (Ii) chain (Cresswell, P. (1994) Annu. Rev. Immunol., 12, 259). The Ii chain shields the peptide binding site of class II molecules during their transport in the cytosol, until they reach an acidic endosomal compartment, where it is cleaved by proteases, leaving only a peptide thereof, termed CLIP, in the binding site. The exchange of CLIP with antigenic peptides is catalysed by another MHC-encoded molecule, termed HLA-DM, in the endosome (Vogt, A. B., et al. (1996) Proc. Natl. Acad. Sci. USA. 93, 9724). The antigenic peptides derive mostly from endocytosed external proteins (Germain, R. N. (1994) Cell, 76, 287).
The nature of interaction between DR molecules and peptides is largely understood. There is one major pocket in the binding site that is critical for the interaction with a hydrophobic anchor residue of the peptide, and additional minor pockets containing polymorphic β chain residues, which confer a degree of allotype-specificity to peptide binding (Stern et al., above; Hammer, J., et al. (1993) J. Exp. Med., 176, 1007; Hammer, J., et al. (1994) Cell. 74,197; Hammer, J., et al. (1994) Proc. Natl. Acad. Sci., USA 91, 4456; Hammer, J., et al. (1995) J. Exp. Med., 180, 2353). The peptide main chain also forms important hydrogen bonds with side chains of certain conserved residues in the binding site, which determine the overall conformation and side chain orientation of the bound peptide (Stern et al., above).
A large body of evidence has demonstrated that susceptibility to many diseases, in particular autoimmune diseases, is strongly associated with specific alleles of the major histocompatibility complex (reviewed in Tiwari, J., and Terasaki, P. (1985), HLA and disease association (New York; Springer Verlag)). Although some class I-associated diseases exist, most autoimmune conditions have been found to be associated with class II alleles. For example, class II alleles DRB1*0101, 0401, 0404, and 0405 occur at increased frequency among rheumatoid arthritis (RA) patients (McMichael, S. J., et al. (1977) Arthritis Rheum., 20, 1037; Stasny, P. (1978) N. Engl. J. Med., 298, 869; Ohta, N., et al. (1982) Hum. Immunol., 5, 123; Schiff, B., et al. (1982) Ann. Rheum. Dis., 41, 403), whereas DRB1*1501 is associated with multiple sclerosis (MS), and the DQ allele combination DQA1*0301/B1*0302 with insulin-dependent diabetes mellitus (IDDM). In RA, altogether >94% of rheumatoid factor positive patients carry one of the susceptibility alleles (Nepom, G. T., et al. (1989) Arthritis, Rheum., 32, 15).
The effect of DRB1 alleles on RA is manifested in different ways: first, the disease association shows ethnic-dependent preference for one or the other allele (Ohta et al., and Schiff et al., above), second, DRB1*0401 is associated with more severe forms of the disease than the other alleles (Lanchbury, J. S., et al. (1991) Hum. Immunol., 32, 56), and third, a gene dosage affect can be observed, in that homozygosity for a susceptibility allele or combinations of two susceptibility alleles confer more severe, chronic forms or juvenile onset of RA (Wordworth, P., et al. (1992) Am. J. Hum. Genet., 51, 585; Nepom, B. S. (1993) Clin. Immunol. Immunopathol., 67, 850). The latter finding indicates that the DRB1 locus can control both initiation and progression of the disease.
The DRB chains encoded by RA-linked DRB1 alleles exhibit polymorphic differences, but all share a stretch of identical, or almost identical amino acid sequence at positions 67-74, known as the “shared epitope” (Nepom et al., (1989) above; Gregersen, P. K., et al. (1987) Arthritis Rheum. 30, 1205). Residues in the shared epitope region contribute to the formation of the α helix on one side of the peptide binding groove (Brown et al., Stern et al., and Dessen et al., above), and are thus expected to influence peptide binding. Indeed, the basic residue Lys or Arg at position (p)71 of RA-associated DR allotypes imparts selectivity on peptide binding by favoring negative and disfavoring positive charge at residue p4 of the displayed peptide, whereas the RA-unlinked allotype DRB1*0402 with acidic residues Asp and Glu at p70 and 71 shows the opposite charge preference at residue p4 of the displayed peptide (Hammer, J., et al. (1995) J. Exp. Med., 181, 1847). Although the autoantigens inducing RA remain unknown, several joint cartilage proteins have peptide sequences which can selectively bind to RA-associated DR molecules due to an acidic residue at p4 (Dessen et al., Hammer et al., (1995) above). These proteins can thus be candidate antigens for an autoimmune response causing RA pathology (Rosloniec, E. F., et al. (1997) J. Exp. Med. 185, 1113). The opposite (positive) charge preference of DRB1-0402 has been shown to confer selective presentation of peptides with a basic residue at p4, derived from desmoglein 3, an autoantigen involved in the 0402-associated autoimmune disease, pemphigus vulgaris (Wucherpfennig, K. W., et al. (1995) Proc. Natl. Acad, Sci. USA, 92, 11935). These data strongly support the hypothesis that selective presentation of autoantigenic peptides by disease-linked MHC allotypes could be the mechanism underlying the genetic association between DRB1 alleles and autoimmune diseases (Todd, J. A., et al. (1988) Science, 240, 1003). The disease process itself is driven by Th cells recognizing such peptides. The activated autoreactive Th cells secrete different pro-inflammatory cytokines, which in turn attract further inflammatory cells to the site, and cause a chronic inflammation in the affected organ.
Of the two classes of MHC molecules, class II is the primary target for immunosuppressive intervention for the following reasons: First, MHC-II molecules activate T helper (Th) cells that are central to immunoregulation, and are responsible for most of the immunopathology in inflammatory diseases. Second, most autoimmune diseases are genetically associated with class II alleles. Third, under normal physicological or non-pathological conditions, MC-II molecules are expressed selectively on cells of the immune system, whereas MHC-I are present on most somatic cells.
Peptide binding to class II (e.g., DR) molecules requires the presence of defined side chains at so-called “anchor positions” of the displayed peptide, which all together form a particular binding motif; however, at non-anchor positions, a variation of side chains is permitted without influence on binding (Hammer et al., (1993, 1994, and 1995), above). This binding mechanism enables the presentation of many different peptides by a given allotype. The side chains at anchor positions interact with specific pockets within the binding site, whereas those at non-anchor positions point outward, and are available for recognition by the TCR of Th cells. It is therefore conceivable that replacement of autoantigenic peptides presented by autoimmune disease-associated MHC molecules by a compound having the same binding motif but being different at non-anchor positions could prevent the activation of autoimmune T cells, and thus interrupt the disease process. The mechanism whereby such a compound would exert its effect is competitive antagonism for the antigen-presenting site. Compounds binding selectively to class II molecules involved in a particular autoimmune disease are therefore expected to interfere specifically with that disease. Additional peptides which bind to MHC molecules and inhibit T cell activation have been disclosed in, for example, International Patent Applications WO 92/02543, WO 93/05011, and WO 95/07707.
A pharmaceutical agent targeting class II MHC molecules would offer several advantages over most available immunosuppressive drugs. First, it would represent a disease mechanism-based intervention, which is expected to interrupt the initial event in the pathogenic cascade. Second, it can be designed to be selective for only a few class II allotypes, i.e., binding with improved affinity to those allotypes associated with disease, leaving the remainder of the antigen presenting system available for protective responses against pathogens, and therefore causing fewer immunocompromising side effects than most immunosuppressive drugs. Third, the methods and compounds could be applied without any specific knowledge of the actual autoantigens causing the disease. Finally, it would be advantageous if such a pharmaceutical agent showed superior stability in certain biological environments. For example, high drug stability in mammalian plasmas such as rat, mouse or human plasma, would be desirable given that many cells of the immune system are found in the blood together with powerful peptide degrading enzymes. High drug stability in rodent plasma, especially rat plasma, is particularly advantageous since most therapeutics are initially tested for efficacy, toxicity, and/or pharmacokinetics in rodent models or systems. Drug stability against Cathepsin degradation is equally desirable since mechanism-based therapeutic intervention requires that pharmaceutical agents targeting class II MHC molecules may be endocytosed and transported within the cell using Cathepsin-containing endosomes before presentation to the MHC II molecule.